Rapid Analysis of Fentanyl and Fentanyl Analogues from Whole Blood Using SPME Coupled to the Microfluidic Open Interface.

Fentanyl and its analogues are potent opioids that pose a significant threat to society. Over the last several years, considerable focus has been on the concerning trend of increasing fentanyl usage among drug users. Fentanyl analogues are mainly synthesized to evade analytical detection or increase their potency; thus, very low concentrations are sufficient to achieve a therapeutic effect. In an effort to help combat the synthetic opioid epidemic, developing targeted mass spectrometric methods for quantifying fentanyl and its analogues at ultralow concentrations is incredibly important. Most methods used to analyze fentanyl and its analogues from whole blood require manual sample preparation protocols (solid-phase extraction or liquid-liquid extraction), followed by chromatographic separation and mass spectrometric detection. The main disadvantages of these methods are the tedious sample preparation workflows, resulting in lengthy analysis times. To mitigate these issues, we present a targeted method capable of analyzing 96 samples containing fentanyl, several fentanyl analogues, and a common fentanyl (analogue) precursor simultaneously in 2.4 min per sample. This is possible by using a high-throughput solid phase microextraction workflow on the Concept96 autosampler followed by manual coupling of solid-phase microextraction fibers to the microfluidic open interface for tandem mass spectrometry analysis. Our quantitative method is capable of extremely sensitive analysis, with limits of quantification ranging from 0.002 to 0.031 ng mL-1 and linearity ranging from 0.010 to 25.0 ng mL-1. The method shows very good reproducibility (1-18%), accuracy (81-100%) of calibration and validation points, and good interday reproducibility (6-15%).

Performance Evaluation of Extraction Coatings with Different Sorbent Particles and Binder Composition.

Binders are critical components used in the preparation of a range of extraction devices, including solid-phase microextraction (SPME) devices. While the main role of a binder is to affix the sorbent particles to the selected support, it is critical to select the optimal binder to ensure that it does not negatively impact the coating's particle sorption capability. This work presents the first comprehensive investigation of the interactions between binders and solid sorbent particles as these interactions can significantly impact the performance of the coating. Specifically, the findings presented herein provide a better understanding of the extraction mechanisms of composite coatings and new rules for predicting the particle adhesion forces and binder distribution in the coating. The influence of binder chemistry on coating performance is investigated by examining a selection of the most used binders, namely, polydimethylsiloxane (PDMS), polyacrylonitrile (PAN), poly(vinylidene difluoride) (PVDF), polytetrafluoroethylene amorphous fluoroplastics (PTFE AF 2400), and polybenzimidazole (PBI). The solid particles (e.g., hydrophilic-lipophilic balanced (HLB) and C18) used in this work were selected for their ability to provide optimal extraction coverage for a broad range of analytes. The results show that PDMS does not change the properties of the solid particles and that the binder occupies a negligible volume due to shrinking after polymerization, resulting in the solid particles making up most of the coating volume. Hence, the coating sorption characteristics correspond closely to the properties of the selected solid particles. On the other hand, the results also showed that PTFE AF 2400 can interact with the active surface of the sorbent, leading to the deactivation of the sorbent particles. Therefore, the extraction performance and permeability coefficients decrease as the size of the penetrant increases, indicating a rigid porous structure. The results of this study can aid in the optimization of SPME devices as they provide reference values that can be used to determine the optimal binder and the sorbent affinity for the targeted compounds. Finally, the present work also provides the broader scientific community with a strategy for investigating the properties of sorbent particle/binder structures and defines the characteristics of a good coating/membrane by analyzing all parameters such as kinetics, thermodynamic equilibria, and morphology.

High-Throughput and Rapid Screening of Drugs of Abuse in Saliva by Multi-Segment Injection Using Solid-Phase Microextraction-Automated Microfluidic Open Interface-Mass Spectrometry.

There is great demand for analytical methods capable of providing high-throughput and rapid screening, especially for anti-doping and clinical point-of-care applications. In this work, automated microfluidic open interface-mass spectrometry (MOI-MS) was used for coupling with high-throughput, automated solid-phase microextraction (SPME) to achieve this objective. The design of the MOI-MS interface provides a continuous and stable electrospray fluid flow to the MS without introducing any bubble, a feature that we exploit to introduce the concept of multi-segment injection for the determination of multiple samples in a single MS run. By eliminating the need to start a new MS run between sample assays, the developed approach provides significantly simplified protocols controlled by programmed software and increased reproducibility. Furthermore, the biocompatible SPME device, which utilizes coating consisting of hydrophilic-lipophilic balanced particles embedded in a polyacrylonitrile (PAN) binder, can be directly used for biological sample analysis, as the PAN acts as both a binder and a matrix-compatible barrier, thus enabling the enrichment of small molecules while eliminating interferences associated with the presence of interfering macromolecules. The above design was employed to develop a fast, quantitative method capable of analyzing drugs of abuse in saliva samples in as little as 75 s per sample. The findings indicate that the developed method provides good analytical performance, with limits of detection ranging between 0.05 and 5 ng/mL for analysis of 16 drugs of abuse, good calibration linear correlation coefficients (R2 ≥ 0.9957), accuracy between 81 and 120%, and excellent precision (RSD% < 13%). Finally, a proof-of-concept experiment was performed to demonstrate the method's suitability for real-time analysis in anti-doping applications.

Polybenzimidazole: a novel, fluorocarbon-free, SPME sorbent binder with good thermal and solvent resistance properties for GC and LC analysis.

A novel solid-phase microextraction (SPME) coating is presented that uses polybenzimidazole (PBI) as a binder to immobilize micro-size sorbent particles onto a support. An evaluation of the developed binder's thermal and solvent desorption capabilities demonstrated its compatibility with both gas and liquid chromatography (GC and LC). The incorporation of hydrophilic-lipophilic balanced (HLB) particles provided optimal extraction coverage for an array of chemically diverse analytes possessing a range of hydrophobicities and molecular weights. The developed binder's performance was assessed by comparing it to a selection of binders commonly used in the literature, including polydimethylsiloxane (PDMS) and polyacrylonitrile (PAN), as well as the more recently developed polyvinylidene fluoride (PVDF) and polytetrafluoroethylene amorphous fluoroplastic (PTFE AF 2400). The results revealed that PBI provides better performance compared to PVDF and PTFE AF 2400 in terms of its environmental impact, while also being convenient for use in coating preparation and offering good matrix compatibility. The thermal analysis revealed that PBI exhibited more than 93% weight retention at 550 °C, which is superior to PVDF's 80.07% weight retention at 393.78 °C. To the best of our knowledge, this work is the first to use PBI as a particle binder in SPME coatings. The PBI coating maintained high extraction efficiencies under extreme conditions with pH values of 3 and 12. The performance of PBI in combination with HLB was assessed by employing it to extract several drugs of abuse and McReynolds compounds for LC and GC analysis, respectively. The results indicated that PBI performs similarly to PAN for LC but is outperformed by PDMS in GC applications with respect to extraction and desorption kinetics. Nonetheless, the thermal and solvent desorption results indicated that PBI can be used for both applications, as it remains stable at temperatures over 350 °C and is stable when solvent desorption is applied.

The Effect of Sorbent Particles in a Binder on the Mass Transfer Kinetics in Separation Media: In Silico Study and Experimental Verification.

Selecting the optimal binder and the sorbent affinity for selected compounds can cause the composite to behave either as an efficient extraction coating, as a permeable membrane, or as an impermeable barrier. If the compound partitions onto the sorbent with high preference, it becomes stationary and the composite behaves as an impermeable barrier, while appropriately optimized affinity will result in effective permeation. To understand this phenomenon, we utilize solid-phase microextraction to characterize the mass transfer attributes of different separation composites. Our results indicate that for strong sorbents, the extraction rate is primarily controlled by the diffusion in the extraction phase rather than the sample matrix, even if it is relatively thin. Low analyte diffusion is caused by the retarding force generated by the partitioning of analytes into the sorbent, as migration through the composite is driven by the unbound form of the compound in the binder. One of the main contributions of this work is that an understanding of the extraction composite parameters that control mass transfer during extraction enables better optimization of binder/sorbent extraction phase composition for a given application. Another contribution of this work shows how a heterogeneous coating model can be simplified into a homogeneous coating model. The developed models enable an enhanced understanding of mass transfer kinetics, and they provide insight into how to optimize the extraction phase parameters for a given method involving sorbent particles in polymeric media, including membranes and paints, in addition to extraction coatings.

Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

Evaluation of a coated blade spray-tandem mass spectrometry assay as a new tool for the determination of immunosuppressive drugs in whole blood.

Immunosuppressive drugs (ISDs) are primarily administered following solid organ transplant or for treatment of a variety of autoimmune conditions. Their principal function is to suppress the activity of the immune system; however, the levels must be carefully monitored due to adverse effects of over- or underadministration. A technology for rapid quantitative screening, named coated blade spray (CBS), was directly coupled to a triple quadrupole mass spectrometer (MS/MS) to measure the concentration of ISDs (i.e., cyclosporine A, tacrolimus, everolimus, sirolimus) in whole blood samples. We evaluated the stability of replicate measurements over a 10-day period (precision), assessed linearity and limit of quantification, and performed a method comparison against a validated clinical immunoassay (Abbott ARCHITECT). Total interday variation of less than 5% for all target compounds at three different concentrations was achieved. The sensitivity of the method was determined as 0.25, 1, 1, and 2.5 ng/mL for everolimus, sirolimus, tacrolimus, and cyclosporine A, respectively. The concentrations of three immunosuppressive drugs in 284 patient samples (i.e., ~ 95 samples of cyclosporine A, tacrolimus, or sirolimus) obtained using the CBS-MS/MS methodology were compared with concentrations previously quantified on an Abbott ARCHITECT immunoassay system. Our analysis demonstrated significant statistical similarities between both methods. The results demonstrate that CBS-MS/MS is a suitable alternative to conventional methodologies for monitoring of ISDs from whole blood in a clinical setting. Graphical abstract.

Measurement of Free Drug Concentration from Biological Tissue by Solid-Phase Microextraction: In Silico and Experimental Study.

In this article, the use of an SPME technique is reported for the first time for direct measurement of free drug concentration in solid tissue. In our investigations, we considered doxorubicin (DOX) spiked in homogenized tissue matrix at transient and equilibrium extraction conditions, with subsequent assessment of obtained experimental results by an in silico approach using mathematical models developed in COMSOL Multyphysics. In silico studies were performed on the basis of transported diluted species (tds) and reaction engineering (re) modules from COMSOL Multiphysics, using the same conditions as those used to attain experimental results. To determine the apparent binding affinity of DOX to the tissue matrix which contains multiple binding species, the experimentally determined binding affinity of DOX with human serum albumin (HSA) was considered to simplify the mathematical calculations. Here, the value of the binding affinity was considered for a single binding site and adjusted by fitting the experimental results with the mathematical model. Bovine lung tissue homogenate was selected as a surrogate matrix, and a biocompatible C-8 commercial SPME fiber was used for extraction of DOX. In total, four mathematical models were herein developed to describe the mass transfer kinetics of solid coatings: in agar gel at static conditions, in PBS solution with agitated conditions, extraction in PBS solution in the presence of an HSA binding matrix, and static extraction in homogenized lung tissue. For all conditions, simulated results were in good agreement with experimental results. The developed mathematical model allows for measurements of free drug concentrations inside the tissue matrix and facilitates calculations of local depletion of DOX by a solid SPME coating. Results of the investigations indicate that local depletion of the free form of DOX, even at the kinetic stage, is negligible for tissue extraction, as the release of the heavily bound analyte (over 99% binding to tissue matrix) is very rapid, thus easily compensating for the loss of the drug to the SPME coating. This indicates that the dissociation rate constant of DOX from lung tissue components is very rapid; therefore, the mass transfer of drug to the fiber coating via free from is very efficient. Our results also indicate that thin coating SPME fibers provide a good way to measure drug distribution after dosing, as extractions via thin coating SPME fibers do not affect the free concentration of the drug, which is responsible for drug distribution in tissue.

Effect of Transport Parameters and Device Geometry on Extraction Kinetics and Efficiency in Direct Immersion Solid-phase Microextraction.

An alternative strategy to increase mass transfer entails geometry optimization of the extraction systems including design of solid-phase microextraction (SPME) probes. In this work, a computational model was employed to elucidate practical aspects such as efficiency and kinetics of extraction by employing several new geometries. Extraction of a model analyte at static conditions with the configurations, such as thin-film, fiber, coated tip, and nanoparticles, was numerically simulated to obtain an in-depth understanding of the advantages and limitations of each geometry in microextraction and exhaustive modes. The attained results associated with the equilibration time dependency on shape were in good agreement with previously reported experimental observations. They demonstrate that the mass-transfer is highly dependent on the size and shape of the coatings and increases with a decrease in size of the devices particularly rapidly below 10 µm caused by radial diffusion effect. Nevertheless, extractions performed using octadecyl-functionalized magnetic nanoparticles demonstrated that higher enrichment factors are achievable with the use of a fewer number of particles in comparison to factors achieved via exhaustive extraction, where a larger number of particles must be employed, confirming theoretical predictions. The conclusions reached are valid for any extraction method. The results obtained herein are very useful toward the design and optimization of future extraction technologies and approaches.

Coupling of Solid-Phase Microextraction Directly to Mass Spectrometry via an Improved Microfluidic Open Interface to Facilitate High-Throughput Determinations.

Mass spectrometry analysis can be performed by introducing samples directly to mass spectrometry, allowing the increase of the analysis throughput; however, some disadvantages of direct-to-mass spectrometry analysis include susceptibility to matrix effects and risk of instrument contamination from inadequate sample preparation. Solid-phase microextraction is one of the most suitable sample preparation methods for direct-to-mass spectrometry analysis, as it offers matrix-compatible coatings which ensure analyte enrichment with minimal or no interference from matrix. One of the ways solid-phase microextraction can be coupled directly to mass spectrometry is via a microfluidic open interface. This manuscript reports improvements made to the initial microfluidic open interface design, where the system components have been simplified to mostly commercially available materials. In addition, the analysis of samples has been automated by implementing software that fully controls the analysis workflow, where the washing procedure is optimized to completely reduce the carryover. Herein, the extraction and desorption time profiles from thin and thick SPME devices was studied where the overall workflow consisted of high-throughput sample preparation of 1.3 min per 96 samples and <1 min per sample instrumental analysis.

Rapid Screening and Quantitation of Drugs of Abuse by Both Positive and Negative Modes via Coated Blade Spray-Mass Spectrometry.

Solid-phase microextraction (SPME)-direct mass spectrometry (MS) has proven to be an efficient tool for the rapid screening and quantitation of target compounds at trace levels. However, it is challenging to perform screening using both positive and negative modes in one analytical run without compromising scanning speed and detection sensitivity. To take advantage of the special geometry of a coated blade spray (CBS) blade, which consists of two flat sides coated with the same SPME coating, we developed a CBS-MS method that enables desorption and ionization to be performed in positive ionization mode on one side of a coated blade and negative ionization mode on the other side of the same blade. By simply flipping the blade 180°, MS analysis in both ionization modes on different sides can be completed in 40 s. Combining this approach with an automated Concept 96-blade-based SPME system allowed analysis for one sample in positive and negative modes to be completed in less than 1 min. The workflow was optimized by using a biocompatible polyacrylonitrile as an undercoating layer and a binder of polyacrylonitrile/hydrophilic-lipophilic balance (HLB) particles, which enabled the rapid analysis of 20 drugs of abuse in saliva samples in both positive and negative modes. The proposed method provided low limits of quantification (between 0.005 and 10 ng/mL), with calibration linear correlation coefficients ⩾ 0.9925, accuracy between 72% and 126%, and relative precision < 15% for three validation points.

Direct Coupling of Bio-SPME to Liquid Electron Ionization-MS/MS via a Modified Microfluidic Open Interface.

We present a modified microfluidic open interface (MOI) for the direct coupling of Bio-SPME to a liquid electron ionization-tandem mass spectrometry (LEI-MS/MS) system as a sensitive technique that can directly analyze biological samples without the need for sample cleanup or chromatographic separations as well as without measurable matrix effects (ME). We selected fentanyl as test compound. The method uses a C18 Bio-SPME fiber by direct immersion (DI) in urine and plasma and the subsequent quick desorption (1 min) in a flow-isolated volume (2.5 µL) filled with an internal standard-acetonitrile solution. The sample is then transferred to an EI source of a triple-quadrupole mass spectrometer via a LEI interface at a nanoscale flow rate. The desorption and analysis procedure requires less than 10 min. Up to 150 samples can be analyzed without observing a performance decline, with fentanyl quantitation at microgram-per-liter levels. The method workflow is extremely dependable, relatively fast, sustainable, and leads to reproducible results that enable the high-throughput screening of various biological samples.

Rapid determination of tacrolimus and sirolimus in whole human blood by direct coupling of solid-phase microextraction to mass spectrometry via microfluidic open interface.

Immunosuppressive drugs are administered to decrease immune system activity (e.g. of patients undergoing solid organ transplant). Concentrations of immunosuppressive drugs (ISDs) in circulating blood must be closely monitored during the period of immunosuppression therapy due to adverse effects that take place when concentration levels fall outside of the very narrow therapeutic concentration range of these drugs. This study presents the rapid determination of four relevant immunosuppressive drugs (tacrolimus, sirolimus, everolimus, and cyclosporine A) in whole human blood by directly coupling solid-phase microextraction to mass spectrometry via the microfluidic open interface (Bio-SPME-MOI-MS/MS). The BioSPME-MOI-MS/MS method offers ≤ 10% imprecision of in-house prepared quality controls over a 10-day period, ≤ 10% imprecision of ClinCal® Recipe calibrators over a three-day period, and single total turnaround time of ∼ 60 min (4.5 min for high throughput). The limits of quantification were determined to be 0.8 ng mL-1 for tacrolimus, 0.7 ng mL-1 sirolimus, 1.0 ng mL-1 for everolimus, and 0.8 ng mL-1 for cyclosporine. The limits of detection were determined to be 0.3 ng mL-1 for tacrolimus, 0.2 ng mL-1 for sirolimus, 0.3 ng mL-1 for everolimus, and 0.3 ng mL-1 for cyclosporine A. The R2 values for all analytes were above 0.9992 with linear dynamic range from 1.0 mL-1 to 50.0 ng mL-1 for tacrolimus, sirolimus, and everolimus while from 2.5 ng mL-1 to 500.0 ng mL-1 for cyclosporine A. To further evaluate the performance of the present method, 95 residual whole blood samples of tacrolimus and sirolimus from patients undergoing immunosuppression therapy were used to compare the Bio-SPME-MOI-MS/MS method against a clinically validated reference method based on chemiluminescent microparticle immunoassay, showing acceptable results. Our results demonstrated that Bio-SPME-MOI-MS/MS can be considered as a suitable alternative to existing methods for the determination of immunosuppressive drugs in whole blood providing faster analysis, better selectivity and sensitivity, and a wider dynamic range than current existing approaches.

Development of thin-film solid-phase microextraction coating and method for determination of artificial sweeteners in surface waters.

A semi-automated and sensitive method was developed for simultaneous determination of the six most consumed artificial sweeteners (AS) in surface waters using thin-film solid-phase microextraction (TF-SPME) and high-performance liquid chromatography (HPLC). A triple quadrupole mass spectrometer and an electrospray ionization source (ESI-MS) run in negative ionization and multiple reaction monitoring modes were employed for instrumental analysis. The TF-SPME method was optimized for the extraction phase, sample pH, desorption solvent, extraction time, and desorption time. In-house-synthetized-hydrophilic-lipophilic balance weak anion exchange (HLB-WAX) particles imbedded within a polyacrylonitrile (PAN) binder were selected as the extraction phase for the thin-film coating due to their cost-effectiveness and enhanced sensitivity for artificial sweeteners. Suitable analytical parameters that include linearity (R2 > 0.9914), recovery > 80%, inter, and intra-reproducibility less than 18% were obtained for the AS compounds studied. The developed method estimated limits of detection (LODs) ranging from 0.004 to 0.038 ng mL-1 The SPME method was successfully applied for the determination of ultra-trace levels of AS in water samples collected from Grand River (Ontario, Canada), downstream of three municipal wastewater treatment plants (WWTPs). Concentrations ranging from 0.03 to 20.3 ng mL-1 were found for the AS compounds studied.